Determination of Piceid and Resveratrol in Spanish Wines Deriving from Monastrell ( Vitis vinifera L. ) Grape Variety

Determination of Piceid and Resveratrol in Spanish WinesDeriving from Monastrell (Vitis vinifera L.) Grape Variety

JUAN F. MORENO-LABANDA ,† RICARDO MALLAVIA ,† LAURA PEÄ REZ-FONS,†

VICTORIA LIZAMA ,‡ DOMINGO SAURA,† AND VICENTE MICOL* ,†

Instituto de Biologıá Molecular y Celular, Universidad Miguel Hernańdez,03202-Elche, Alicante, Spain, and Departamento de Tecnologıá Agroalimentaria, Escuela Politećnica

Superior de Orihuela, Universidad Miguel Hernańdez, 03312-Orihuela, Alicante, Spain

The presence of stilbenes in wine is becoming an important issue due to their claimed relation to alow incidence in coronary diseases and their increasing implication as cancer chemopreventive andneuroprotective agents. Total resveratrol content, quantified as glucoside and aglycone forms ofresveratrol, has been determined in a survey of 45 Monastrell monovarietal Spanish red wine types(around 135 wine samples), belonging to Alicante and Bullas appellations. The average betweenratio glucoside/aglycone forms of resveratrol in these wines was considerably high, ranging from 82to 91% of resveratrol in its glycosidic form. This characteristic was observed in a high percentage ofthe studied wines, which were made under different winemaking procedures, and from differentvintages (1995-2002). In addition, wines made using macerative fermentations with double amountof solid parts (“doble pasta”) reached the highest levels of total stilbene content expressed asresveratrol equivalent, i.e., 30 mg/L (average of 18.8 mg/L). It can be concluded that high resveratrolglucoside concentration and low free isomer content can be considered characteristics of the Monastrellvariety, as it happens to red wines deriving from other varieties grown at warm climates. This fact,also observed for other French and Portuguese red varieties, might play an important role in foodhabits involving these types of wines.

KEYWORDS: Resveratrol; piceid; red wine; Monastrell ; HPLC

INTRODUCTION

Phytoalexines from theVitaceaeseem to be confined to agroup of compounds belonging to the stilbene family (1), whoseskeleton is based on the 1,2-diphenylethylene structure (Figure1). Resveratrol (3,5,4′-trihydroxystilbene) and piceid (3,5,4′-trihydroxystilbene-3-â-D-glucoside) (Figure 1) are two of themajor stilbene phytoalexins produced byVitaceae (1-4).Mulberries, grapes, and wine are considered the most importanthuman dietary sources of stilbenes (5). trans-Resveratrol issynthesized especially in skin and leaves (1, 3, 6, 7), where itwas first detected in 1976 (5). Another stilbene (Figure 1) suchas piceatannol glucoside (3,5,3′,4′-tetrahydroxystilbene-3-â-D-glucoside), also named astringin, has been also found inVitisVinifera cell cultures and wine (8, 9). These compounds arepresent in wine in their cis and trans forms depending on factorssuch as UV radiation and winemaking process. There are otherstilbene derivatives such as diglucosides, methylated, or poly-meric forms, but these do not seem to be present in wine.

In the past decade, there has been a great interest in thepresence oftrans-resveratrol in wine due to its supposed protective effects against cardiovascular metabolism related

diseases (10-12), as derived from several epidemiologicalstudies (13, 14). The mechanism of resveratrol’s cardiovascularbenefits has not been totally elucidated, but it has been attributedto its antioxidant (9, 15) and anticoagulative properties (16).trans-Resveratrol has also exhibited chemopreventive activityagainst carcinogenesis (17, 18). In addition, resveratrol has

* To whom correspondence should be addressed. Dr. Vicente Micol,Instituto de Biologıá Molecular y Celular, Universidad Miguel Hernań-dez. Edificio Torregaitań, Avda. de la Universidad s/n 03202-ELCHE(Alicante), Spain. Tel.:+34-96-6658430. Fax:+34-96-6658758. E-mail:[email protected].

† Instituto de Biologıá Molecular y Celular.‡ Escuela Politećnica Superior de Orihuela.

Figure 1. Chemical structures of the stilbenes frequently found in wines.1 and 4, trans- and cis-resveratrol; 2 and 5, trans- and cis-piceid; 3 trans-astringin.

5396 J. Agric. Food Chem. 2004, 52, 5396−5403

10.1021/jf049521m CCC: $27.50 © 2004 American Chemical SocietyPublished on Web 07/31/2004

shown antioangiogenic activity (19, 20). Recently, severalphenolic compounds from plants, resveratrol among them, havebeen demonstrated to exert neuroprotection against the oxidativestress derived from chronic ethanol administration (21) or nitricoxide (NO) production in cultured neurons (22, 23). Consider-able interest has thus been focused on foodstuffs related toMediterranean Aliment Culture lifestyle, where olive oil, grape,and wine consumptions seem to play an important role (24).

The presence of stilbenes in wine has been widely analyzed,to find profiles related to varieties, although further investiga-tions are needed in this direction. Stilbenes concentration inwines vary depending on multiple factors such as grape variety,fungal infections, winemaking procedures, and climatologicalconditions (25, 26) from values under 0.1 mg/L up to∼20 mg/L(8, 26). Moreover, the glucoside forms of resveratrol are sup-posed to be cleaved to its aglycon form, resveratrol, by hydro-lysis during the winemaking process (3, 8, 26). Red wines us-ually contain higher stilbene concentrations than rose or whitewines, probably due to a more prolonged skin contact duringfermentation and their higher overall phenolic content (27-29). Resveratrol concentration was reported to be below or near1 mg/L in studies done on several American wines (27, 28)and wines from Burgundy (29). Low resveratrol or piceidconcentrations were also found in a survey of Japanese wines(30), except for those deriving from Pinot noir or Merlot. Highervalues fortrans-resveratrol, up to 6-7 mg/L, were reported byMattivi et al. in several Italian wines deriving from Cabernet-Sauvignon or Merlot (25). A larger survey was performed oncommercial wines from different regions by Goldberg et al.(31-33). In the latter study, the highest levels of averageresveratrol (trans plus cis isomers) and average piceid werefound for Burgundy (5.4 and 5.1 mg/L, respectively), Bordeaux(7.9 and 2.2 mg/L) and Niagara, Canada (5.4 and 8.6 mg/L),although a considerable variability was also found. A surveyon several Spanish wines done by Lamuela-Ravento´s et al. (34)reported total average resveratrol values of 9.3 and 9.1 for Pinotnoir and Merlot, respectively. Furthermore, values of total res-veratrol equiv up to 17.5 mg/L with a content oftrans-resveratrolglucoside of 11 mg/L were found in red wines deriving fromMourVedre variety (35).

Regarding red wines, certain cultivars such as Pinot Noir orMerlot seem to contain higher stilbene concentrations thanCabernet-Sauvignon, Muscat, Concord, Grenache, or Tempra-nillo (30, 34, 36, 37). Moreover, the region of growth and itstemperature may significantly influence the stilbene concentra-tion (2, 38). Most wines, such as Burgundy, Bordeaux, Pinotnoir, or Merlot (26, 34), exhibit profiles with higher amount ofresveratrol in its free form than that of glycosidic derivatives.On the contrary, some wines from warm and dry regions (Spain,Italy, Portugal and South America) seem to have low concentra-tion of free isomers and relatively high glucoside concentrations(26, 38-40).

In this work, a total of 45 monovarietal wine types (around135 wine bottles) deriving fromMonastrellgrapes and belongingto two different appellations of Spain, that is, Alicante and Bullas(Murcia), have been analyzed by HPLC, to contribute to theknowledge on stilbene content in relation to grape variety. Thisstudy has included regular wines and wines made underparticular winemaking practices such as oak-aged, organic or“doble pasta” wines, from 1995 to 2002 vintages. The averagecontent of the glycoside and aglycone forms of trans-resveratroland its ratio was also determined. In addition, this is the firststudy in whichMonastrellvariety has been exhaustively studied.

MATERIALS AND METHODS

Chemicals.trans-Resveratrol andâ-glucosidase (12.4 units/mg) werepurchased from Sigma-Aldrich Chemical Co. (St. Louis, MO).trans-Piceid was purified by preparative HPLC, as described below, from aPolygonum cuspidatumroot aqueous extract (Hu Zhang variety)obtained from DYNAFYT and distributed by NOVASAN S.A (Madrid,Spain). Methanol, acetonitrile, trifluoroacetic acid, methanol-d4, andacetone-d6, were purchased from Merck (Darmstadt, Germany). Allsolvents employed were liquid chromatography grade.

Wine Samples.trans-Piceid (trans-resveratrol glucoside),cis-piceid,trans-resveratrol, andcis-resveratrol were determined for 45 differentred wine types, that is, 135 wine samples (750 mL bottles), many ofthem from commercial origin, belonging to two different appellations(“Denominaciones de Origen”, D. O.) of Spain, Alicante and Bullas(Murcia). Red wine samples were monovarietal, pure monovarietal orblends (blended red wine contained 80% or higherMonastrellgrapevinevariety) from different vintages covering from 1995 to 2002.

Sample Preparation for HPLC Analysis.Samples were analyzedby direct HPLC injection, after filtration through 0.2-µm Whatmaninorganic Anodisc 13 membrane filters from Whatman InternationalLTD (Maidstone, England). When needed, wine samples were subjectedto hydrolysis digestion withâ-glucosidase, according to a previouslypublished method (2, 38). Briefly, 1 mL of wine was neutralized withNaOH until approximately pH 6.0, then 4 mg ofâ-glucosidase wasadded, and digestion was allowed for 18 h at 25°C under darkness.

Standards. Isolation and purification oftrans-piceid was done byusing a preparative HPLC Knauer-Merck Wellchrom system equippedwith two K-1800 pumps (250 mL/min) for binary elution, a dynamicblend camera, a manual injector, a K-2600 UV detector, and a fractioncollector FC K-6 from Bu¨chi (Flawil, Switzerland). A 12-mL sampleof Polygonum cuspidatumroot aqueous extract (110 g/100 mL) wascentrifuged, filtered through a 0.45-mm nylon filter, and injected intopreparative HPLC. Solvents and conditions used for preparative HPLCwere the same as those described for the analytical one, as describedlater on this section, but using a flow adapted to a bigger size column(39 mL/min). UV detection was set at 280 and 306 nm. A LiChrospherRP-18 column (250 mm× 25 mm, 15 µm) from MERCK wasemployed for the chromatographic separation of the root aqueous extractof Polygonum cuspidatum. trans-Piceid was the most abundantcompound at 306 nm (data not shown for briefness). Fractions derivingfrom preparative isolation and containing only pure compound, aschecked by analytical HPLC, were combined and vacuum concentrated.A yield of approximately 50 mg oftrans-piceid was obtained. Purifiedtrans-piceid was further analyzed by analytical HPLC and showed aUV spectrum nearly identical totrans-resveratrol, as previouslydescribed by other authors (32, 41), showing a broad absorption bandbetween 290 and 330 nm with its maximum at 320 nm. To confirm itsidentity, trans-piceid was digested withâ-glucosidase as describedearlier, and puretrans-resveratrol was obtained. In addition,1H and13C NMR spectral data, which are shown later within this section, wereidentical to those previously described (3, 42). cis-Resveratrol andcis-piceid were obtained by UV irradiation of diluted solutions of theirrespective trans isomers as described elsewhere (32, 41) and were usedto construct calibration curves obtained from data at 285 nm forquantitation ofcis-isomers in wines.cis-Piceid andcis-resveratrolstandards were handled in darkness due to their photosensitivity (43).

HPLC Analysis and Quantification. Separation and quantificationof stilbenes were done by HPLC using a method modified from thatone described by Dalluge et al. (44). trans-Piceid andtrans-resveratrolwere quantified using their respective pure standards. Concentrationsof the trans isomers were determined using the external standard methodand their response factors were deduced from their respective calibrationcurves. A Merck-Hitachi LaChrom system equipped with a D-7100quaternary pump, D-7455 diode-array detector, D-7485 fluorescencedetector, and L-7650 column oven was used. An analytical LiChrospher100 RP-18 column (250 mm× 4 mm, 5µm) from Merck was utilizedand protected by a guard column of the same material. Separation wasperformed at a flow rate of 1 mL/min with a mobile phase composedof (A) H2O + 0.05% TFA and (B) 60:40 MeOH-ACN+ 0.05% TFA.Samples (25µL) of each wine were injected into the HPLC system

Piceid and Resveratrol in Spanish Monastrell Wines J. Agric. Food Chem., Vol. 52, No. 17, 2004 5397

after filtration as described above. The multigradient solvent systemwas as follows: 0-5 min, from 10% B to 15% B; 5-40 min, from15% B to 35% B; 40-55 min, from 35% B to 10% B. The columnwas equilibrated with starting conditions for 20 min after each analysis.Diode-array detection was used, and oven temperature was 35°C. Datawere obtained from triplicate analyses on up to three wine bottles foreach studied wine type. Relative standard deviation was always under3% of the values. Cis isomer of resveratrol was found in negligibleamounts (less than 0.05 mg/L) in all cases, therefore, data for thiscompound were not shown.

Nuclear Magnetic Resonance Spectroscopy.Nuclear magneticresonance spectra fortrans-resveratrol glucoside identification werecollected on a Bruker AVANCE 500 spectrometer and probe HD 5mm TXI 13C Z. The spectra were collected at 296 K in methanol-d4or acetone-d6 as deuterated solvents, and the resonance of each methylgroup of the solvents was used as reference for shift tabulated values(45). NMR experimental parameters corresponding totrans-resveratrolglucoside structure, which were confirmed with those previouslypublished by other authors (3, 42), were as follows:

1H NMR (500 MHz, CD6CO, ppm): δ 7.42 (d, 2H,J ) 8.25 Hz,H2′ and H6′), 7.08 (d, 1H,J ) 16.4 Hz, H7 or HR), 6.91 (d, 1H,J )16.2 Hz, H8 or Hâ), 6.84 (d, 2H,J ) 8.2 Hz, H3′ and H5′), 6.81 (broadsignal, 1H, H2), 6.67 (bs, 1H, H6), 6.46 (bs, 1H, H4), 4.93 (d,J ) 7.6Hz, H1′′), 3.91 (bs, 1H, H6′′), 3.72 (bs, 1H, H6′′), 3.54-3.43 (4H, m,H2′′, H3′′, H4′′, H5′′).

1H NMR (500 MHz, CD3OD, ppm): δ 7.37 (d, 2H,J ) 8.6 Hz,H2′ and H6′), 7.02 (d, 1H,J ) 16.2 Hz, H7 or HR), 6.85 (d, 1H,J )16.3 Hz, H8 or Hâ), 6.79 (t, 1H,J ) 1.6 Hz, H2), 6.77 (d, 2H,J ) 8.6Hz, H3′ and H5′), 6.62 (t, 1H,J ) 1.6 Hz, H6), 6.45 (t, 1H,J ) 2.15

Hz, H4), 4.89 (bs+ water signal, H1′′), 3.94-3.92, 3.73-3.69 (bs,2H, 2 × H6′′), 3.48-3.45 (4H, m, H2′′, H3′′, H4′′, H5′′).

13C NMR (125 MHz, CD3OD, ppm): δ 160.47 (C3), 159.57 (C5),158.46 (C4′), 141.42 (C1), 130.31 (C1′), 129.97 (C8 or Câ), 128.91(C2′ and C6′), 126.65 (C7 or CR), 116.48 (C2′ and C6′), 108.33 (C6),106.99 (C2), 104.07 (C4), 102.40 (C1′′ or C anomeric), 78.24 (C5′′),78.04 (C3′′), 74.95 (C2′′), 71.47(C4′′), 62.58 (C6′′).

RESULTS AND DISCUSSION

Monastrellgrape variety, also known asMourVedre in South-ern France, orMataro in California andCataluna, is a nativered variety from the Spanish Mediterranean coast.Monastrellgrapes exhibit thick-skinned berries, which allow them to thrivevigorously in warm and arid climates. Wines produced fromthis variety tend to be high in alcohol and tannins and havedistinctive balsamic, blackberries, and mineral flavors, especiallywhen young. This is the major variety in several Spanish DOs(Appellations) such as Almansa, Valencia, Jumilla, Yecla, Ali-cante, and Bullas. In the last yearsMonastrell-based wines haveconsiderably increased their value supposing an attractive highquality alternative to other well-established varieties in Spain.

The unequivocal structure oftrans-resveratrol-â-D-glucopy-ranoside was elucidated by monodimensional (1H and13C NMR)and bidimensional HSQC (1H and13C). The monodimensionalspectra in both deuterated solvents were coincident with thosepreviously published by other authors (3, 42). Assignation ofNMR signals by HSQC in acetone-d6 is shown inFigure 2.

Figure 2. 2D Proton-Carbon HSQC NMR spectrum of trans-resveratrol-â-D-glucopyranoside in acetone-d6.

5398 J. Agric. Food Chem., Vol. 52, No. 17, 2004 Moreno-Labanda et al.

The trans configuration of the isolated compound was confirmedby the large coupling constant (J ) 16.3 Hz). Data of relevantsignals, in each deuterated solvent, such as the stilbene systemand the anomeric carbon of the glucoside moiety were collectedand confirmed (see bold assignments in NMR materials section).

To contribute to the characterization of the stilbenes profilein Monastrell wines, the levels of some of these compoundswere determined in wines produced under different winemakingprocesses.Table 1 shows the content of three stilbenesdetermined in 21 youngMonastrellwines of vintages from 1997to 2002 belonging to Alicante and Bullas appellations (Spain).The majority of wines analyzed showed higher levels oftrans-piceid than oftrans-resveratrol, reaching quite high concentra-tions of trans-piceid ranging from 15 to 17 mg/L (wines 3, 4,and 9), as compared to other red varieties such as Pinot noir orMerlot, which showed around 4 mg/L (34), or red wines fromSpain, France or Canada, which showed levels around 3-7mg/L (32). In most cases studied in this work, levels of cisisomers were much lower than their respectivetrans-isomers,with cis-resveratrol levels being almost negligible (<0.05 mg/

L). The molar ratio glycoside/aglycone forms of resveratrolyielded an average value of 4.63, meaning that around 82% ofresveratrol was in its glucosidic form. Vintages 2001 (wines9-13), 2002 (wine 14) and some wines of vintage 2000 (wines3, 4, and 20) showed the highest level of total resveratrol withinthese wine groups (defined as the sum of cis and trans forms ofpiceid and resveratrol) mainly due to their high content intrans-piceid. Although some young wines deriving from Alicante DOexhibited total resveratrol amount a little higher than those ofBullas appellation, their average total resveratrol values werequite similar, 6.84 and 6.62 mg/L, respectively.

Piceid and resveratrol levels for a group of oak-agedMonastrellwines of vintages from 1995 to 2001 are shown inTable 2 (“crianza”, 24 months oak barrel+ bottle-aged withat least 12 months oak barrel-aged; “reserva”, 36 months oakbarrel+ bottle-aged with at least 12 months oak barrel-aged).In this case, wines from vintages 2000 (wine 27) and 2001 (wine28) also exhibited the highest content of total equivalentresveratrol, i.e., 10.6-12.3 mg/L. A previous study focused onthe relationship between aging process and resveratrol content

Table 1. Stilbene Content (mg/L) for Young Red Wines Deriving from Monastrell Variety Grown in the Spanish Mediterranean Coast

winetype vintage

apellation(D. O.)

trans-piceid(mg/L)

cis-piceid(mg/L)

trans-resveratrol(mg/L)

ratioa

piceid/resveratrol

totalresveratrol

(mg/L)b

1 1999 Alicante 4.20 ndc 0.08 32.32 2.532 1999 Alicante 1.31 nd 1.14 0.67 1.913 2000 Alicante 15.38 nd 1.69 5.31 10.684 2000 Alicante 16.30 nd 1.53 6.23 11.065 2000 Alicante 7.19 nd 3.66 1.15 7.866 2000 Alicante 6.24 nd 1.83 1.99 5.477 2000 Alicante 1.19 nd 1.55 0.45 2.258 2000 Alicante 6.56 nd tracesd 1.929 2001 Alicante 17.09 nd 2.60 3.84 12.59

10 2001 Alicante 11.80 nd 2.13 3.23 9.0311 2001 Alicante 13.04 nd 3.16 2.41 10.7812 2001 Alicante 11.72 nd traces 3.4313 2001 Alicante 7.44 nd 1.05 4.15 5.4014 2002 Alicante 7.34 7.24 2.34 3.65 10.8615 1997 Bullas 6.08 nd 1.09 3.27 7.0416 1998 Bullas 9.33 nd 1.59 3.43 6.9117 1998 Bullas 9.59 nd 1.53 3.67 5.8218 1999 Bullas 8.65 nd 1.86 2.73 4.6419 1999 Bullas 6.48 5.34 1.75 3.95 7.1420 2000 Bullas 7.52 nd 1.43 3.08 8.6621 2000 Bullas 7.52 nd 1.75 2.51 6.15

total avg 8.66 6.29 1.78 4.63 6.77

a Expressed as a molar ratio b Expressed as resveratrol equivalent (aglycone form) c nd, not detected d traces (<0.05 mg/L)

Table 2. Stilbene Content (mg/L) for Oak-Aged Red WinesDeriving from Monastrell Variety Grown in the Spanish Mediterranean Coast

winetype vintage

apellation(D. O.)

trans-piceid(mg/L)

cis-piceid(mg/L)

trans-resveratrol(mg/L)

ratioa

piceid/resveratrototal

resveratrol(mg/L)b

22 1997, crc Alicante 7.18 4.56 2.56 2.68 9.4223 1997, cr Alicante 7.56 ndd 0.22 20.24 4.6424 1998, cr Alicante 7.78 nd 1.29 3.52 5.8425 1998, cr Alicante 6.95 nd 0.18 22.15 4.2526 1999, cr Alicante 6.03 2.23 tracese 2.4227 2000, cr Alicante 17.24 nd 2.20 4.59 12.2828 2001, cr Alicante 11.83 5.36 0.57 17.66 10.6229 1997, rec Alicante 10.21 nd 0.32 18.45 6.3030 1995, cr Bullas 9.06 nd 0.97 5.48 6.2631 1996, re Bullas 9.87 nd 1.44 4.00 7.2132 1998, cr Bullas 6.89 nd 0.83 4.88 4.8533 1998, cr Bullas 14.67 nd 1.80 4.75 10.38

total avg 9.61 4.05 1.13 9.85 7.04

a Expressed as a molar ratio b Expressed as resveratrol equiv (aglycone form) c crianza, cr; reserva, re d nd, not detected e traces (<0.05 mg/L)


performed on Ontario red wines showed important losses inboth trans- and cisisomers of resveratrol (46). However, theaverage of total resveratrol found in the analysis done in thiswork encompassing a total of twelve agedMonastrell wineswas close to 7 mg/L, similar to the value observed for youngwines in Table 1. Average ratio glucoside/aglycone for agedwines was a little bit higher, yielding more than 90% ofresveratrol in its glucoside form. In general, wine-aging processfor Monastrellwines did not seem to diminish the content ofthe glycosidic form of resveratrol in comparison to nonagedwines. According to our results, a previous study showed thatwines made from Mourve´dre variety conserved both resveratroland piceid and presented similar total resveratrol concentrationcontents regardless of their age (35).

A group of unusual wines, such as sweet, organic, or doblepasta wines, all made fromMonastrellgrapes and deriving fromAlicante DO, were also analyzed, and their resveratrol contentis shown inTable 3. Sweet redMonastrellwines (wines 34-36) presented low or undetectable levels of the stilbenesanalyzed in this work, and only sweet “Fondilloń” showedsignificant levels oftrans-piceid. Wines made from organicgrapes (wines 37-40) showed similar or even higher levels ofresveratrol toMonastrell wines made with regularly culturedgrapes. AMonastrellwine from vintage 2002 and made from

organic grapes (wine 39) yielded one of the highest contents intotal resveratrol, that is, 20.59 mg/L, a value remarkably highconsidering that this wine was not subjected to special macera-tion process. In contrast, an aged organic wine (wine 40) showedvery low levels of total resveratrol compared to nonaged ones,maybe due to the lower stability of these compounds under thelow SO2 concentrations and higher pH used in such wines (47).A group of particular wines called doble-pasta made fromMonastrell grapes were also analyzed (wines 41-45). Doblepasta wines are obtained by the addition of solid parts left fromrose winemaking process to a regular maceration-fermentationof a red wine, so maceration is made in the presence of doubleamount of solid material. This process yields wines with highcolor intensity and an alcoholic content as high as 16%.Monastrell doble pasta wines showed the highest level ofstilbenes of all the wines studied. It is noteworthy that a doblepasta wine (wine 45) reached a considerably high content intrans-piceid (almost 30 mg/L), yielding a remarkable value oftotal resveratrol quantified as resveratrol equiv (i.e., 30.88 mg/L). Figure 3 shows the HPLC chromatogram corresponding tothis particular doble pastaMonastrell wine (2002) in whichtrans-piceid was the major compound observed at 306 nm. Theaverage of total resveratrol for doble pastaMonastrell winesreached a value of 18.80 mg/L, higher than average values found

Table 3. Stilbene Content (mg/L) for Organic, Sweet and “doble Pasta” Red Wines Deriving from Monastrell Variety Grown in the SpanishMediterranean Coast

wine typewine

sample vintageapellation

(D. O.)trans-piceid

(mg/L)cis-piceid

(mg/L)trans-resveratrol

(mg/L)ratioa

piceid/resveratrol

totalresveratrol

(mg/L)b

34 sweet Fondillon 1944 Alicante 4.51 nd tracese 2.6435 sweet Fondillon 1980 Alicante 6.00 nd traces 3.5136 sweet 2001 Alicante ndd nd nd nd37 organic 2000 Alicante 5.83 4.55 traces 3.0438 organic 2001 Alicante 11.89 3.72 1.36 6.69 10.4939 organic 2002 Alicante 16.88 11.40 4.05 4.08 20.5940 aged organic, crc 2000 Alicante 4.15 3.49 traces 2.2541 doble pasta 2001 Alicante 22.08 10.18 2.89 6.53 21.7542 doble pasta 2001 Alicante 17.69 5.96 2.15 6.44 15.9743 doble pasta 2001 Alicante 11.90 4.52 2.98 3.22 12.5844 doble pasta 2001 Alicante 13.07 7.31 0.92 12.93 12.8445 doble pasta 2002 Alicante 29.24 14.80 5.13 5.02 30.88

total avg 13.02 7.33 2.78 6.41 12.41

a Expressed as a molar ratio. b Expressed as resveratrol equiv (aglycone form). c crianza: cr. d nd, not detected. e traces (<0.05 mg/L).

Figure 3. HPLC chromatogram corresponding to doble pasta Monastrell wine type 45 obtained at 306 nm, showing a high concentration of trans-piceid.Peak 1, trans-piceid; peak 2, cis-piceid; peak 3, trans-resveratrol.


for the majority of previously studied red wines, as reviewedin the Introduction (30, 34, 35, 37, 39, 48, 49). An increase ofthe stilbene content up to 10-fold has also been observed byother authors in wines made using long macerative fermenta-tions, which allow a prolonged skin contact (47, 50). Consider-ing the results obtained from the 45Monastrellwines studied,the average of total equiv resveratrol was around 8.25 mg/L, avalue which is close to what it has been found also in Spanishwines for otherhigh stilbene leVel varieties such as Pinot Noiror Merlot (i.e., 9.3 and 9.1 mg/L, respectively) (34).

A previous study on transfer and stability of stilbenes duringfermentation and aging in aMourVedre variety (35), a varietyidentical to Monastrell but geographically adapted to SouthFrance, has also shown a higher content in piceid compared toresveratrol (67 vs 33%, respectively) but more similar valuesof cis andtrans-piceid were obtained than in our case. Thesedata are in agreement with our results and support the statementthat a high piceid/resveratrol ratio is an intrinsic characteristicof Monastrellvariety, which behaves independently from growthlocation and condition, according to which it had been suggestedfor other red varieties such as Pinot Noir (8, 34).

Although other authors have previously found relatively highvalues of piceid in several red varieties, including Spanish wines(32, 34), the values found in this study forMonastrell winesare superior to those found in most cases and are comparableto the maximum levels found in a study done on monovarietalPortuguese wines (8). The latter study found significant averagevalues oftrans-piceid (i.e., 11.8 and 13 mg/L in Portugueseand French wines, respectively). In our study, normal fermenta-tion Monastrell wines yielded average values oftrans-piceidof 8-10 mg/L (Tables 1 and 2). Furthermore, double macera-tive fermentation wines (doble pasta)Monastrellwines reachedalmost 30 mg/L oftrans-piceid, with a mean value of 18.8 mg/L. Thus, high piceid content can be associated to this variety.It also has to be considered that a significant percentage of thewines studied (38%) showed values oftrans-piceid over 10mg/L and that 50% of the wines showed values of total stilbenehigher than 7 mg/L. In addition, in allMonastrell winesanalyzed, 82-91% of the stilbenes were in their glycosidic form.Considering that other stilbenoids such as astringin or viniferinswere not determined in this study, it is assumed that total stilbenecontent ofMonastrellwines could be even higher. The resultspresented in this work show thatMonastrell wines exhibitmarkedly high levels oftrans-piceid and low level of freetrans-resveratrol. Since this fact was observed along several vintagesand a diversity of winemaking processes, it can be postulatedthat this feature may suppose a phenotypical varietal character,as it has been postulated by other authors for other varietiesgrown at warm climates (26, 38-40).

An estimation of the total stilbene intake fromMonastrellwine (8), and considering a regular consumption of 200 mL/day ofMonastrellwine, would mean a daily intake of stilbenes(calculated on the basis of the average value of total resveratrol)of approximately 1.65 mg/day/individual, and 3.8 mg/day/individual for people only drinking doble pasta wines. Consider-ing that resveratrol bioavailability by oral administration in ratsis around 40% (51), resveratrol peak plasma concentrationachieved after ingestion of these wines would probably be withinthe lowµM range, which might be increased after a prolongedconsumption. These values are not too far from those concentra-tions at which resveratrol has demonstrated its biological activity(52, 53). There are increasing evidences of the existence ofâ-glucosidase activity in human small intestine (54, 55) soresveratrol glucoside may be deglycosylated and converted into

bioavailable resveratrol during digestion. It has been also shownthat trans-piceid shares similar biological activities with itsaglycone form,trans-resveratrol (56-58). Nevertheless, it isstill unclear if piceid’s biological activity is exerted by itself orthrough resveratrol conversion; thus, further pharmacokineticstudies are needed.

ABBREVIATIONS USED

DO, appellation (denominacioń de origen); HPLC, high-performance liquid chromatography; HSQC, heteronuclearsingle quantum coherence; NMR, nuclear magnetic resonance;UV, ultraviolet light.

ACKNOWLEDGMENT

We thank Raquel Navarro for her HPLC assistance.

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Received for review March 24, 2004. Revised manuscript received June18, 2004. Accepted June 20, 2004. This investigation was supported byGrants QADVSC1999-0052, QADVSC2000-70, and QADVSC2001-174from the Instituto Valenciano de Calidad Agroalimentaria, Consellerıade Agricultura, Pesca y Alimentacion, Generalitat Valenciana (V. M.),and Funds from CRDO Alicante (Consejo Regulador de la Denomi-nacion de Origen Vinos de Alicante, Spain) and CRDO Bullas(ConsejoRegulador Denominacioń de Origen “Bullas”, Murcia, Spain).

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Documents

Determination of Piceid and Resveratrol in Spanish Wines Deriving from Monastrell ( Vitis vinifera L. ) Grape Variety